Novel approaches for chemoprevention of breast cancer are desirable because many risk factors associated with this disease are not easily modifiable. Moreover, some of the currently available chemopreventive options targeted against breast cancer are sub-optimal. Needless to emphasize that breast cancer continues to be a leading cause of cancer-related death in women worldwide despite tremendous advances towards targeted therapies. Ongoing objective of this research project is to develop a non-endocrine strategy for chemoprevention of breast cancer using cruciferous vegetable constituent Benzyl Isothiocyanate (BITC). Research objectives of the current funded grant were nearly fully achieved with notable publications. For example, we demonstrated that BITC administration in the diet confers significant protection against mammary cancer in a transgenic mouse model (MMTV-neu) without any signs of overt toxicity. We are extremely excited with our more recent published as well as unpublished observations underpinning novel actions of BITC potentially contributing to its chemopreventive effect, including inhibition of epithelial-mesenchymal transition (EMT) in vitro and in vivo (published) and suppression of self-renewal of breast cancer stem cells (bCSC) in vitro (unpublished observations). We also found that BITC treatment activates Notch signaling, which is a positive regulator of EMT as well as CSC self-renewal. Thus it is only logical to experimentally test whether Notch activation by BITC has negative impact on its chemopreventive response. At the same time, expression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) is significantly downregulated by BITC treatment. Because uPAR overexpression is sufficient to drive both EMT and stemness in breast cancer cells, further investigation of the role of uPA/uPAR in BITC-mediated inhibition of EMT and bCSC self-renewal is equally meritorious. Hypothesis: Research design in the present renewal application logically builds upon these exciting and novel observations to test a stimulating hypothesis that mammary cancer chemoprevention by BITC is mediated by inhibition of the uPA/uPAR system leading to suppression of EMT and self-renewal of bCSC, which may be amenable to augmentation by pharmacological suppression of Notch using a ?-secretase inhibitor. Specific Aims: Proposed research utilizes relevant cellular and in vivo animal models of breast cancer to determine: (1) the impact of Notch activation by BITC on its effects contributing to mammary cancer chemoprevention; (2) the contribution of uPA/uPAR suppression in BITC-mediated inhibition of EMT; and (3) obtain in vivo evidence for efficacy of BITC against bCSC self-renewal and to study the role of uPA/uPAR system in this response. Translational Impact of the Proposed Research: Studies conducted thus far provide compelling preclinical evidence for chemopreventive efficacy of BITC against breast cancer, but efficient translation of these findings into a clinical setting is dependent on a full understanding of the molecular pharmacology of BITC driving its chemopreventive responses. Clinical trial design without a full appreciation of the molecular pharmacology of BITC may be sub-optimal. Defining innovation of the current application includes translational merit of the proposed research as well as novel research directions. For example, intrinsic value of the studies proposed in Aim 1 resides in potential rational design of a combination regimen involving BITC and a ?-secretase inhibitor to achieve even greater chemopreventive efficacy. Aim 2 may identify biomarkers of BITC response (e.g., uPA and uPAR) potentially useful in future clinical investigations. Discovery of biomarker(s) predictive of BITC response is an equally meritorious objective because cancer incidence is too rigorous of an end point for malignancies with long latency such as breast cancer. Likewise, experimental validation of the in vivo efficacy of BITC against bCSC may (in future) lead to novel BITC- based regimens for clinical management of breast cancer because the existing mechanistic model stipulates critical role for bCSC not only in cancer development and progression but also in resistance to therapy. PUBLIC HEALTH RELEVANCE: Breast cancer is a leading cause of cancer-related death in women worldwide. Novel approaches for chemoprevention of breast cancer are clinically attractive. Long-term objective of this research project is to develop a safe and inexpensive but effective regimen for chemoprevention of breast cancer using cruciferous vegetable constituent benzyl isothiocyanate (BITC). Research conducted in the previous funding period provided preclinical evidence for efficacy of BITC against breast cancer, but efficient translation of these observations into a clinical setting depends on full appreciation of the molecular pharmacology of BITC. Clinical trial design without a full appreciation of the molecular pharmacology of BITC may be sub-optimal. Defining innovation of the current application includes translational merit of the proposed research as well as novel research directions.